The commercial market for recombinant protein biopharmaceuticals is expanding rapidly as various biotechnology and pharmaceutical companies develop and test biologically active proteins. The emerging field of proteomics will likely provide protein targets useful for drug development, thereby enabling the market for recombinant protein biopharmaceuticals to continue its expansion.
Currently, proteins are utilized in a variety of diagnostic and therapeutic applications. For example, one protein used in a diagnostic application is the enzyme glucose oxidase, which is used in glucose assays. The hormone insulin is an example of a protein utilized in therapeutic applications. However, proteins are particularly sensitive to certain environmental conditions and may not be stable at elevated temperatures, including physiological temperature of 37° C., in non-optimal aqueous solvent systems, or in organic solvent systems. Protein stability may also be affected by pH and buffer conditions and exposure to shear forces or other physical forces.
The stability of a protein refers to both its conformational stability, which is reflected in the protein's three-dimensional structure, and its chemical stability, which refers to the chemical composition of the protein's constituent amino acids. Protein instability can result in a marked decrease or complete loss of a protein's biological activity. Deleterious stresses such as organic solvents, extremes of pH, high temperatures, and/or dehydration (drying) can affect both the conformational and chemical stability of a protein. Chemical instability can result from (a) deamidation of the amino acids residues asparagine or glutamine, (b) oxidation of cysteine or methionine amino acid residues, or (c) cleavage at any of the peptide amide linkages of the protein. Examples of conformational instability include aggregation (fibrillation), precipitation, and subunit dissociation.
Because an inactive protein is useless, and in some cases deleterious, for most diagnostic and therapeutic applications, there is a need for a means by which proteins can be stabilized both in dry form and in solution. It is known in the art that proteins can be stabilized in solution by the addition of soluble excipients that stabilize the monomeric, correctly folded protein conformation. Disaccharides such as trehalose, sucrose, or lactose, and surface active agents such as phospholipids, Tween, and Triton are examples of excipients useful for stabilizing proteins. These stabilizers must be used in non-toxic levels because in the case of therapeutic proteins, the stabilizers are necessarily administered to the patient with the protein.
U.S. Pat. No. 5,834,273 issued to Futatsugi et al. on Nov. 10, 1998 provides a heat and protease resistant enzyme with improved storage stability. This enzyme is modified with a polysaccharide, polyamino acid, or synthetic polymer having a plurality of carboxyl groups by means of a crosslinking agent capable of binding both carboxyl groups and amino groups.
U.S. Pat. No. 5,736,625 issued to Callstrom et al. on Apr. 7, 1998 discloses a method for preparing water soluble, saccharide-linked protein polymer conjugates that stabilize the protein in a hostile environment. The claimed method includes covalently binding the polymer to the protein through at least three linkers, each linker having three or more hydroxyl groups. The protein is conjugated at lysines or arginines.
U.S. Pat. No. 5,691,154 issued to Callstrom et al. on Nov. 25, 1997 provides an enzyme linked immunoassay in which the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate includes the enzyme which is linked to the polymer at multiple points through saccharide linker groups.
U.S. Pat. No. 5,612,053 issued to Baichwal et al. on Mar. 18, 1997 discloses a powder formulation which includes cohesive composites of particles containing a medicament and a controlled release carrier which includes one or more polysaccharide gums of natural origin.
U.S. Pat. No. 5,492,821 issued to Callstrom et al. on Feb. 20, 1996 discloses water soluble protein polymer conjugates in which proteins linked to an acrylic polymer at multiple points by means of saccharide linker groups. These conjugates are also stable in hostile environments.
U.S. Pat. No. 5,128,143 issued to Baichwal et al. on Jul. 7, 1992 provides a slow release pharmaceutical excipient of an inert diluent and a hydrophilic material including xanthan gum and a galactomannan gum capable of cross-linking the xanthan gum in the presence of aqueous solutions.
Ispas-Szabo et al. demonstrated that the ability of starch tablets to swell and release low molecular weight drugs could be controlled by the degree that the starch was cross-linked. No data related to protein stabilization was presented. Carbohydrate Research 323, 163-175 (2000).
Artursson et al. demonstrated that proteins could be incorporated into polyacryl starch microparticles. One incorporated protein, the enzyme carbonic anhydrase, retained a low amount of activity at temperatures where the free protein had no activity (e.g., >70° C.). At lower temperatures (e.g., <65° C.), however, the free enzyme was more stable than the enzyme incorporated into the microparticles. Journal of Pharmaceutical Sciences 73, 1507-1513 (1984).
Gliko-Kabir et al. demonstrated that the swelling of lyophilized guar gum powder in gastric or intestinal buffer could be reduced from approximately 100 fold to approximately 5 fold if the guar was crosslinked with glutaraldehyde. No data concerning protein stabilization was presented. Pharmaceutical Research 15, 1019-1025 (1998).
Bauman et. al. demonstrated that carrageenan gum stabilized the enzyme cholinesterase against heat when the enzyme was dried on a urethane foam sheet with 8% starch. Analytical Biochemistry 19, 587-592, (1967).
Many of the methods that are known to stabilize proteins, require that the protein be covalently attached to a solid support or covalently substituted with a stabilizing molecule. Covalent modification is not always practical for proteins in solutions, thus there is a need for a protein stabilization system that does not require covalent modification of the protein.
The typical method of administering therapeutic proteins to a patient or test subject is by means of needle-based injections. Currently, many pharmaceutical and drug delivery companies are seeking to develop alternative systems for the delivery of therapeutic proteins. These alternative systems are expected to require fewer dosings and to allow for more effective control over the rate of protein release in the body.
One alternative drug delivery system known in the art includes the formulation of the protein in a biodegradable polymer matrix. The polymer (e.g., poly(lactic-co-glycolic acid)) can be formulated as an injectable or respirable microparticle. Alternately, the protein can be formulated in a temperature sensitive polymer that is liquid at room temperature but solidifies at 37° C. after injection into a patient. In both cases, the polymer systems are developed for sustained release of protein over time; however, the stability of the protein during the release period is difficult to maintain and generally less than 50% of the total protein load can be delivered. Additionally, the delivery of the protein is not uniform, but rather occurs with a rapid initial burst which is followed by a much slower rate of sustained protein release.
A second type of known delivery system includes an implanted pump such as an osmotic pump. In this system, a suspension of protein in a water miscible organic solvent is continuously delivered to the patient or test subject through an orifice in the osmotic pump implant. However, use of this system may prove problematic because it is often difficult to suspend a high protein load in the organic solvent, and only some proteins are stable to prolonged incubation under the required non-aqueous or mixed organic-aqueous conditions.
Thus, given the current state of the art, there is a need for compositions and methods that effectively stabilize a variety of proteins in various chemical and physical environments, and that are compatible with a variety of drug delivery systems.